Project Summary Our results show that the peptide neurotensin (NT) and its high affinity receptor (NTR1) play a critical role in the pathophysiology of both colonic inflammation and colon cancer by activating expression of proinflammatory, proliferative and anti-apoptotic genes. Gene expression is negatively regulated by microRNAs, leading to mRNA degradation, and/or inhibition of translation into protein. MiRs are also important regulators of Inflammatory Bowel Disease (IBD). We recently reported that NT/NTR1 interactions in human colonocytes differentially regulate expression of microRNAs that affect colon tumorigenesis through stimulation of miRNA feedback networks involving inflammatory and anti-apoptotic pathways. This is the first evidence in the literature supporting the notion that neuropeptides modulate micorRNA expression in colonic epithelial cells and that this interaction is functionally correlated with modulation of a GI disease. Here we will test the novel hypothesis that the NTR1 - regulated microRNAs miR-210 and miR-133a play an important role in the pathophysiology of IBD-like colitis by activating pathways linked to inflammatory signaling and NTR1 internalization. The following aims will address these hypotheses. Aim 1 will determine the importance of miR- 210 in NT-associated proinflammatory responses in human colonic epithelial cells and examine the upstream and downstream signaling pathways involved in this response. Aim 2 will assess the importance of miR-133a in NTR1 trafficking and its role as a link of proinflammatory responses to receptor trafficking in human colonic epithelial cells and study the mechanisms eliciting this response. Use of human disease samples will confirm the IBD relevance of the findings in aims 1 and 2. Aim 3 will determine the functional significance of the relationship of NT and NTR1 to miRNAs-210 and 133a in models of colitis. Studies in this aim include use of NT, and NTR1 deficient mice in models of colitis that will help verify NT-associated miR-210-driven signaling targets and angiogenesis and NT-linked miR-133a-associated receptor trafficking. These studies should advance our understanding on the pathogenesis of IBD and define the role and mechanisms of NTR1- microRNA signaling in this group of diseases.